荧光Real-time PCR鉴定鸡胸骨总RNA质量

为了更准确地鉴定提取鸡骨总RNA的质量,试验分别用核酸蛋白检测仪、1%琼脂糖凝胶电泳和荧光Real-time PCR检测评价3种不同的方法提取成年鸡胸骨总RNA的质量。结果显示,荧光Real-time PCR可更好地鉴定提取的骨总RNA的质量。用核酸蛋白检测仪、1%琼脂糖凝胶电泳能粗略检测RNA的纯度和完整性,但不能反映提取过程中是否引起基因不均一的减少,所检测的纯度也不能精确反映RNA的反转录效率。     

肝细胞癌患者外周血HSF1 基因表达的检测及应用价值

目的:建立检测HSF1 mRNA的real-time PCR的方法,了解肝细胞癌患者外周血中HSF1的表达水平及其与各临床病理特征之间的关系。方法:利用real-time PCR的方法检测20例肝细胞癌患者及20例正常人群外周血中HSF1 mRNA的表达量。结果:肝细胞癌患者外周血中的HSF1 mRNA表达量显著高于正常人群(P<0.05);肝细胞癌患者外周血中HSF1 mRNA的表达水平在不同性别、肿瘤大小、门静脉侵犯情况、HbsAg水平及AFP水平的患者中的差异无统计学意义(P>0.05);在不同病理分化程度、TNM分期的患者中的差异有统计学意义(P<0.05)。结论:real-time PCR技术可以成功检测外周血中HSF1 mRNA的表达量,HSF1可能与肝细胞癌的发生发展密切相关。     

重叠PCR 法构建XLRS1 三种不同类型突变体

目的:构建X连锁视网膜劈裂(XLRS)三种不同类型突变体(点突变、缺失突变、插入突变),以便进一步构建不同类型的突变细胞模型。方法:根据NCBI gene数据库中XLRS1基因的CDS设计三对不同类型的引物,使用重叠PCR法获得突变片段,然后克隆入载体pLJM1-EGFP。结果:经测序验证,成功构建了三种不同类型突变体。结论:使用重叠PCR法构建三种不同类型突变体,为进一步研究XLRS1不同类型突变的致病机制奠定了基础。     

粪肠球菌生物被膜形成早期相关基因的表达研究

目的:以粪肠球菌为研究对象,探讨粪肠球菌基因srtA(转肽酶A编码基因)、esp(肠球菌表面蛋白)与粪肠球菌生物被膜形成早期的相关性。方法:用逆转录PCR与实时荧光定量PCR方法对生物被膜和浮游菌组细菌srtA、esp两种与生物被膜形成早期相关的基因其表达进行检测,并进行统计学分析。结果:srtA、esp基因与粪肠球生物被膜菌早期形成密切相关。生物被膜菌组srtA、esp表达量分别是浮游菌组的7.9与13.5倍。结论:srtA、esp基因与粪肠球生物被膜菌形成早期密切相关,可能是生物被膜早期形成的上调因子。     

PCR enrichment techniques to identify the diet of predators

The increasing sensitivity of PCR has meant that in the last two decades PCR has emerged as a major tool in diet studies, enabling us to refine our understanding of trophic links and to elucidate the diets of predators whose prey is as yet uncharacterized. The achievements and methods of PCR-based diet studies have been reviewed several times, but here we review an important development in the field: the use of PCR enrichment techniques to promote the amplification of prey DNA over that of the predator. We first discuss the success of using group-specific primers either in parallel single reactions or in multiplex reactions. We then concentrate on the more recent use of PCR enrichment techniques such as restriction enzyme digests, peptide nucleic acid clamping, DNA blocking and laser capture microdissection. We also survey the vast literature on enrichment techniques in clinical biology, to ascertain the pitfalls of enrichment techniques and what refinements have yielded some highly sensitive methods. We find that while there are several new approaches to enrichment, peptide nucleic acid clamping and DNA blocking are generally sufficient techniques for the characterization of diets of predators and highlight the most important considerations of the approach.     

Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.     

A molecular revision of the taxonomic status of mermithid parasites of black flies from Quebec (Canada)

The four currently recognized mermithid (Nematoda) species parasitizing black flies (Diptera: Simuliidae) from Northeast America were distinguished using discriminatory PCR primers aimed at COI and 18S rDNA. Isomermis wisconsinensis, Gastromermis viridis and Mesomermis camdenensis were easily differentiated using either genomic target, even for juvenile mermithids damaged beyond morphological recognition. However, specimens from Mesomermis flumenalis being identical in external morphology and producing a unique-sized PCR product were classified by sequence data into four clearly distinguished molecular variants. This quartet was made of two winter and two summer ‘physiological variants’, including one which also belonged to, but diverged early from the rest of the Mesomermis genus. Combining the multiplex PCR and sequencing approaches allowed for the characterization of a multiple parasitism which simultaneously implicated I. wisconsinensis and two M. flumenalis variants. With another instance where parasites were identified by morphology only, this is the first report of black fly parasitism by multiple mermithid species. A phylogenetic tree built by combining our sequences to previous GenBank entries likely indicates a monophyletic origin for the mermithid family, but also suggests that differentiation between parasite genera sometimes occurred before the evolutionary emergence of the actual host group.     

Interaction with 7SL RNA but not with HIV-1 genomic RNA or P bodies is required for APOBEC3F virion packaging

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F), like APOBEC3G, has broad antiviral activity against diverse retroelements, including Vif-deficient human immunodeficiency virus (HIV)-1. Its antiviral functions are known to rely on its virion encapsidation and be suppressed by HIV-1 Vif, which recruits Cullin5-based E3 ubiquitin ligases. However, the factors that mediate A3F virion packaging have not yet been identified. In this study, we demonstrate that A3F specifically interacts with cellular signal recognition particle (SRP) RNA (7SL RNA), which is selectively packaged into HIV-1 virions. Efficient packaging of 7SL RNA as well as A3F was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. Reducing 7SL RNA packaging by overexpression of SRP19 protein inhibited A3F virion packaging. Although A3F has been shown to interact with P bodies and viral genomic RNA, our data indicated that P bodies and HIV-1 genomic RNA were not required for A3F packaging. Thus, in addition to its well-known function in SRPs, 7SL RNA, which is encapsidated into diverse retroviruses, also participates in the innate antiviral function of host cytidine deaminases.